The long term goals of this project are to further elucidate the molecular mechanisms of multidrug resistance utilizing cell lines which demonstrate clinically relevant levels of drug resistance. Our approach focuses on understanding the biochemical and genetic basis for movement of mammalian cell from low to high level resistance and from single to multidrug resistance. Such data will help in designing new approaches to combatting drug resistance. This proposal has three specific aims. First, to determine if alterations in intracellular pH are responsible for the decreased uptake of drug noted in the resistant cell lines. This will be done using the fluorescent dye BCECF-AM. Second we will determine if alterations in topoisomerase II are a component of early resistance. This work will include studies of topoisomerase II catalytic activity, ability to form drug stabilized cleavage complexes, protein levels and localization. We will also examine using a novel assay how alterations in topoisomerase II effect drug distribution among various chromatin fractions released from a micrococcal nuclease digestion. Finally, we will clone and sequence the altered topoisomerase II allele.